Legionnaires' disease is an acute pneumonic illness caused by Gram-negative bacilli of the genus Legionella, the most common of which is Legionella pneumophila. 
Legionnaires' disease is initiated by inhalation, and probably microaspiration, of Legionella bacteria into the lungs. Although Legionella bacteria are ubiquitous in our environment, they rarely cause disease. A number of factors must occur simultaneously before legionnaires' disease is possible. These factors include the presence of virulent strains in an environmental site; a means for dissemination of the bacteria, such as by aerosolization; and proper environmental conditions allowing the survival and inhalation of an infectious dose of the bacteria by a susceptible host.
Water contaminated with a sufficient concentration of virulent Legionella bacteria can be aerosolized by water-cooled heat rejection devices such as air conditioning cooling towers, whirlpool spas, shower heads, water misters, and the like. Once the bacteria enter the lung, they are phagocytosed by alveolar macrophages and then grow intracellularly. The Legionella bacteria produce virulence factors that enhance phagocytosis. After sufficient intracellular growth, the bacteria kill the macrophage, escape into the extracellular environment and are then rephagocytosed by other macrophages. Within a few days after initial infection, the bacterial concentration in the lung increases considerably. The resulting infiltration of the alveoli by neutrophils, additional macrophages and erythrocytes results in capillary leakage and edema and the chemokines and cytokines released by the macrophages help trigger a severe inflammatory response, which may be fatal.
More than 49 different Legionella species, encompassing 70 serogroups, have been described since its first discovery in 1977, 20 of which have been reported to infect humans. L. pneumophila contains at least 16 different serogroups. L. pneumophila serogroup 1 caused the 1976 Philadelphia outbreak and is the cause of 70% to 90% of all cases of legionnaires' disease. In the major outbreaks such as those originating in Bovenkarspel, The Netherlands in 1999, in Barrow-in-Furness in Cumbria, England in 2002 and near Harnes in Pas-de-Calais, France in 2003/2004, serogroup 1 strains could be detected in the majority of the patients.
L. pneumophila serogroup 1 can be further divided into multiple subtypes using a variety of serologic, other phenotypic and genetic methods. One particular subtype of L. pneumophila serogroup 1 causes the majority of cases of legionnaires' disease due to L. pneumophila, and 85% of the cases due to L. pneumophila serogroup 1; this subtype is distinguished by its reactivity with a particular monoclonal antibody, and it is variously termed Pontiac, the Joly monoclonal type 2 (MAb2), or the Dresden monoclonal type 3/1 (MAb 3/1) monoclonal subtype.
Most clinical microbiology laboratories are capable of identifying Legionella bacteria to the genus level by detection of their typical colony morphology, Gram stain appearance, and various other standard microbiology identification techniques. Identification of L. pneumophila serogroup 1, the most common clinical isolate can be accomplished by sophisticated clinical microbiology laboratories using relative simple serologic testing. However, it should be stressed that serogroup 1 strains are not the only virulent serotypes
Identification of other L. pneumophila serogroups, and other Legionella species, is often much more difficult. This is because these bacteria are relatively inert in the use of commonly tested biochemical substrates, and they require sophisticated phenotypic, serologic and molecular testing. Reference laboratory-based phenotypic testing of bacteria, including determination of fatty-acids and ubiquinones and protein electrophoresis, can often be used to identify the bacteria. Definitive identification is based on both immunologic detection of surface antigens and bacterial DNA sequencing. Also, DNA typing by pulsed field electrophoresis of DNA restriction fragments is useful for identification purposes.
Diagnosis of the infecting agent from sample material (such as water that is suspected of contamination, or of clinical samples) on selective media is currently the most reliable means of diagnosis. However, cultivation is slow. Direct fluorescent antibody (DFA) stains for the visualization of Legionella species in clinical specimens are commercially available for a limited number of species. Assays for the detection of L. pneumophila serogroup 1 antigen in urine have a sensitivity of 70% and a specificity of nearly 100%. Also, an immunochromatographic assay for the rapid qualitative detection of L. pneumophila serogroup 1 antigen (Legionella NOW; Binax, Portland, Me.) in urine specimens has become available that uses rabbit anti-L. pneumophila serogroup 1 antibody as the capture component and rabbit anti-L. pneumophila serogroup 1 antibody conjugated to colloidal gold as the detection component. The assay provides a test result in 15 min and is intended to aid in the presumptive diagnosis of Legionnaires' disease caused by L. pneumophila serogroup 1 in conjunction with culture and other methods. Preliminary performance data for the immunochromatographic assay report a sensitivity of 95% and a specificity of 95%. Thus, so far, commercially available tests for Legionella urinary antigen detect only L. pneumophila serogroup 1, while the specificity of the assays cannot prevent the occurrence of false positive and false negative reactions.
DNA probe techniques, which produce fewer false positive reactions then immunological detection methods, may be used to detect the presence of one or more multiple Legionella species. However, a drawback of such DNA methods is that they cannot differentiate between virulent and non-virulent strains, presumably because the virulence trait is multi-genic. Further, they are not able to adequately identify Legionella strains if more than one strain is present in the sample.
Following severe outbreaks, many national authorities have implemented legislation and water quality standards for water supplies and/or codes of practice for management and operation of cooling towers and warm water storage facilities. Such standards and codes require frequent monitoring of drinking water distribution systems and swimming-pool water facilities, and upon exceeding a certain number of Legionella bacteria per liter, rigorous measures are taken, such as closure and evacuation of hotels, sports facilities or nursing homes. Most experts however, consider that a large number of Legionella bacteria detected are harmless and non-virulent. However, there is at present no assay system available, except for the tedious assay involving culturing of individual bacteria, which takes about 7 days, to correctly identify possible harmful strains if more than one bacterial strain is present in the sample. The availability of a test that is capable of reliably detecting DNA markers from a specific single Legionella strain would be highly favorable.
It is an object of the present invention to provide for a method capable of identifying multiple strains of L. pneumophila, and especially to discriminate between serogroup 1 strains, strains within this serogroup 1 and strains from other serogroups.